Method of using monoclonal antibodies to cytokeratin fragments

ABSTRACT

Provided is a method for reproducible production of cytokeratin antigen/immunogen. Cytokeratins from whole carcinoma cells are purified by preparative SDS-PAGE. Bands corresponding to cytokeratins 8, 18, and 19 are eluted from the gel, and these cytokeratins are digested to produce fragments in the size range of 10-50 Kd. The invention also relates to use of these fragments as immunogens for the production of antibodies. Furthermore, the invention relates to an immunochemical test kit to detect cancer of epithelial origin in body fluids. The kit comprises cytokeratin fragments produced by the method of the invention and antibodies to these fragments.

This application is a continuation of application Ser. No. 08/030,100,filed on Mar. 23, 1993, the entire contents of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to tumour markers, more specificallycytokeratins, for cancer cells of epithelial origin. According to theinvention a method for reproducible production of cytokeratinantigen/immunogen is provided.

There is a large need to be able to detect and diagnose cancer in anearly stage before the patient has developed an inoperable tumour ormetastases. Furthermore, it is desirable to be able to localize thetumour for localized treatment or prior to surgery.

An example of localized cancer treatment is where the tumour is killedwith antibodies coupled to, for example, cytotoxin or radioactiveisotopes according to known methods. These substances targeted againstthe tumour provide increased killing of tumour cells or an increase ofthe concentration of cytotoxin in the tumour and thereby decrease theside effects of the conventional cytostatic treatment.

From immunohistological and immunocytological tests it is known thatcertain carcinomas, i.e. tumour tissue of epithelial origin, containtumour markers in the form of cytokeratins of different kinds. There are19 different characterized cytokeratins, all being built of proteins.These cytokeratins form so called intermediate filament in the cells.The cytokeratin pair 8 and 18 are very frequent in simple epithelia.

The present invention is based on the discovery that the insolubleintracellular cytokeratins are released and fragmented in tumourtissues, whereby a large fraction of the cytokeratins becomes soluble.The soluble cytokeratin fragments leak out to surrounding body fluids,such as blood, urine, ascites and pleura.

DESCRIPTION OF RELATED ART

In U.S. Pat. No. 4,774,620 cytokeratin fragments released in tissueculture medium of MCF-7 carcinoma cells are used to produce monoclonalantibodies. This method is not reproducible and is very unspecific sincethe actual tumour marker is not exactly known. Because unspecific cellmaterial also is used as reference in tests, the latter becomeunreliable as regards quantification and specificity.

In EP A1 337 057 cytokeratins are chromatographically purified andenzyme digested to obtain "the alpha helical center portions thereof"which in turn are chromatographically purified and used as antigens inimmunological tests and for production of monoclonal antibodies,respectively. Here the antigen/immunogen is obtained in a reproducibleway. These known methods are sufficient for detecting cytokeratinfragments in body fluids and for characterizing to which cellcategory/type a tumour belongs to. However, the monoclonal antibodiesobtained according to EP A1 337 057 require an initial solubilization ofthe sample to be tested.

SUMMARY OF THE INVENTION

Thus, there still exists a need to be able to detect and localizecarcinomas in vivo and treat cancer patients with monoclonal antibodytherapy. Also, there is a need of more simple and sensitive tests thanthose of the prior art for determining whether a tumour is progressiveor not and following up monoclonal antibody therapy to evaluate thetreatment. The present invention fulfils the above needs.

The cytokeratin fragments produced according to the present inventioncause a stronger immune response of an animal injected therewith, andtherefore higher reactivity and specificity of the antibodies, thanthose of the prior art. The present invention also gives a moreappropriate reaction with several different sizes of cytokeratinfragments, although with retained specificity.

Below the invention will be described in a non limiting way. Theabbreviations used are given at the end of the description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the specificity and reactivity of 15different monoclonal antibodies of the present invention in ELISA forcytokeratin 8 (left bar), and 18 (right bar).

FIG. 2 is a bar graph showing the specificity and reactivity of 15different monoclonal antibodies of the present invention againstcytokeratin 19 in ELISA, as in FIG. 1. Six clones, having an absorbanceabove 100, regarded as being reactive with cytokeratin 19.

FIG. 3 shows a Western blot on a nitrocellulose filter after SDS-PAGE ofpurified cytokeratin 8 fragmented with chymotrypsin according to thepresent invention.

FIG. 4 shows a Western blot on nitrocellulose after SDS-PAGE of purifiedcytokeratin 18 fragmented with chymotrypsin.

FIG. 5 shows a Western blot on nitrocellulose after SDS-PAGE of purifiedcytokeratin 19 fragmented with chymotrypsin.

FIG. 6 shows, by ELISA, that the serum from apparently healthy personsgives a low response to monoclonal antibodies of the present invention,with a very low spread. The majority (95%) of the population falls under0.9 ng/ml. For serum from the majority (54%) of cancer patients, thevalues are significantly higher.

FIG. 7 shows cultivated tumour cells (MCF-7) which have been fixed withmethanol (to open up the cell membrane) and thereafter incubated withone of the monoclonal antibodies (M1) according to the presentinvention, followed by a secondary antibody with FITC coupled thereto.

FIG. 8 shows an immunohistological section of an adenoma carcinoma ofthe colon.

FIG. 9 shows mice inoculated with DU 145 cells and with developedtumours.

FIG. 10 shows scintigraphy of the mice of FIG. 9 showing the in vivolocalizations of the tumours.

FIG. 11 shows an immunoscintigraphy image of a human being.

FIG. 12 shows the sensitivity of immunological tests performed withfragments of cytokeratins 8 and 18 according to the present invention.This figure shows a dose-response curve in absorbance at 490 nm and theprecision profile of an ELISA test using fragments of cytokeratins 8 and18 according to the present invention as standard/reference material andthe monoclonals (6D7 and 3F3) coated to microtiter plates. Thesensitivity calculated as average value+2 SD is as low as 0.1 ng/ml,which proves to be a very high sensitivity. In this Figure, two valueshave been measured at each concentration and each value plotted as anopen square. Two slightly differing values at a given concentrationappear as overlapping squares.

FIG. 13 shows the sensitivity of immunological tests performed with thefragments of cytokeratins 8 and 18 according to the present invention.Results similar to those shown in FIG. 12 are obtained in an IRMA testusing monoclonal antibodies 6D7 and 3F3. The data measures the countsper minute (cpm).

DETAILED DESCRIPTION OF THE INVENTION

Production of the cytokeratins (CYK) 8, 18 and 19

Cytoskeleton of the tumour cell line MCF-7, ATCC no. HTB 22, a humanbreast cancer adenocarcinoma cell line from pleura fluid, was preparedaccording to known methods, i.e. Geisler N., Weber K., Eur. J. Biochem.111, 425-433, 1980. Other tumour cell lines of epithelial origin canalso be used, for example DU 145 (ATCC no. HTB 81), HeLa (ATCC no. CCL2) etc.

This material was purified by preparative SDS-PAGE, wherein themolecular weights in relation to molecular weight references showedabout 53, 45 and 41 Kd for the cytokeratins 8, 18 and 19, respectively.This pattern has previously been shown for these cytokeratins. (Moll R.,Franke W. W., Schiller D. L. et al, Cell 31, 11-24, 1982).

The bands corresponding to the cytokeratins 8, 18 and 19 were cut outfrom the gel and extracted with 0.1% SDS, PBS pH 7.5 according toconventional technique. A sample thereof was run on an analyticalSDS-PAGE to verify the purity; the result was single bands for each ofthe cytokeratins 8, 18 and 19 having the same mobility as the threebands in the initial material. This verifies that the cytokeratins 8, 18and 19 have been purified and maintained their size.

Fragmentation of the purified cytokeratins 8, 18 and 19

According to the present invention, the fragmentation of cytokeratinscan be made either enzymatically or chemically, with for examplechymotrypsin, V8 protease, pepsin, TPCK trypsine, BrCN, partialhydrolysis, BNPS Saktole, etc., provided that a reproducible cleavagepattern is obtained with molecular weights between about 10 to about 50Kd of the cytokeratin fragments. Because of the purification methodaccording to the present invention, the fragments do not aggregate, butare kept in a monomeric form. This is very important when the fragmentsare to be used for antibody production as aggregates tend to causemasking of the epitopes.

In a preferred embodiment of the invention the purified cytokeratins 8,18 and 19 are fragmented, each separately, in a controllable manner bychymotrypsin, with a weight ratio enzyme:cytokeratin of about1:50-1:1000, the preferred weight ratios being 1:400 (CYK 8), 1:100 (CYK18) and 1:75 (CYK 19). The activity of the enzyme is 40-60 units per mgof protein. The concentration of cytokeratins is 0.2 mg/ml. Acytokeratin solution and an enzyme solution are each preincubatedseparately for 5 min. in a 37° C. waterbath. Thereafter the enzymesolution is added to the cytokeratin solution and the mixture isincubated for an additional 5 min. in a 37° C. waterbath. The digestionis stopped by adding a small volume of 20% SDS solution and a finalincubation for 5 min. in a 95° C. waterbath. The final SDS concentrationwill be 2%.

Following the digestion an optional purification of the fragments isperformed. The fragments are purified on a preparative SDS-PAGE andafter the electrophoresis a thin gel strip is cut out from the gel andstained for a short time in Coomassie Blue solution. Followingdestaining the position of the different fragments is determined andthese parts of the remaining gel are cut out from the gel and extractedwith 0.1% SDS in phosphate buffer, pH 7.5. A rerun in an analyticalSDS-PAGE verifies the purity.

Fragments having sizes in the range 10-50 Kd are eluted as above. Also,this optional SDS-PAGE of the fragments allows selection of specificfragments. A possible application of this is to immunize mammals withonly one or a few cytokeratin fragments. The special fragment(s) givingespecially reactive antibodies from an animal injected therewith aresequenced. On the basis of this sequence, a synthetic nucleotidesequence is made being inserted into a vector, e.g. a plasmid, andcloned in e.g. a bacterium for large scale production of the desiredfragment.

Production of monoclonal antibodies directed against fragmentedcytokeratins 8, 18 and 19

Cytokeratin fragments in the size range of 10-50 Kd from CYK 8, 18 and19, each purified separately, were dialyzed against 0.1% SDS in PB andthereafter they were immunized in Balb/c mice according to standardprocedures, 10 μg fragments per mouse in FCA (s.c.), 10 μg per mouse inFIA, followed by 1 μg per mouse in FIA twice, all with an interval ofone week. The mice were boostered 3 times with one day in between beforeinjection.

72 hours after the last boost, lymphocytes from the spleen of the micewere collected and fused with Sp2/0 myeloma cells in the relation 1:1.Of course, it is possible to use other mouse species and myeloma cells.The hybridoma cells were allowed to grow and were cloned according toknown methods twice with a dilution technique. One cell per well wasestablished by checking the microtiterplates under a microscope. Thehybridoma cells were allowed to grow, stabilized and established. Atotal of 15 clones was established producing monoclonal antibodieshaving the specificity and reactivity described below.

Test of the specificty and reactivity of the obtained monoclonals

A. ELISA TEST WITH WHOLE CYTOKERATINS 8, 18 AND 19 AS ANTIGEN

When testing the obtained monoclonal antibodies in ELISA, againstpurified cytokeratin 8, 18 and 19 separately coupled to microtiterplates by adsorption at pH 9.0, it appears that some monoclonals havespecificty against one, two or all three cytokeratins, but withdifferent reactivity.

The result of testing the 15 above obtained clones at a concentration 10ng/ml of the monoclonals, is shown in FIGS. 1 and 2, where thereactivity is expressed as absorbance units (490 nm) on the y-axis. FIG.1 is a bar chart showing the specificity and reactivity of the 15different monoclonal antibodies in ELISA for cytokeratin 8 (left bar),and 18 (right bar).

The concentration of the antigens, i.e. cytokeratin 8, 18.and 19 coupledon the plates was each 0.3 μg/ml. The primary incubation with themonoclonals was only for 1 hour at RT. Therefore, only the monoclonalswith the highest reactivity were selected. The secondary antibodies wereanti-mouse IgG antibodies (i.e. Dakopatts Code P260) coupled to HRP in adilution of 1:1000, incubated for another 2 hours RT, and after additionof the substrate o-phenylendiamine, the absorbance at 490 nm was readaccording to conventional ELISA technique. As appears from FIG. 1, eightclones have the highest reactivity against cytokeratin 8 while the otherseven have the highest reactivity against cytokeratin 18.

The bar chart according to FIG. 2 shows the specificity and reactivityof the 15 different monoclonals against cytokeratin 19 in ELISA (asabove). Five clones are regarded as being reactive with cytokeratin 19.

As appears from the figures, there is cross reactivity between thedifferent cytokeratins and this is probably due to the large amino acidhomologies that are present between cytokeratins 8, 18 and 19. (Leube R.E., Bosch F. X., Romano V. et al, Differentiation 33, 69-85, 1986;Romano V., Hatzfeld M., Magin T. M., et al. Differentiation 30, 244-253,1986; and Bader B. L., Magin T. M., Hatzfeld M., Franke W. W. EMBO J. 5,1865-1875, 1986.)

B. WESTERN BLOT WITH FRAGMENTED CYTOKERATIN 8, 18 AND 19 AS ANTIGEN

Western blot of cytokeratin fragments was performed in that thecytokeratins, enzyme digested as above, were run in SDS-PAGE andthereafter were blotted onto nitrocellulose filters according to knownmethods. In this way it was shown that the monoclonal antibodiesidentified the majority of the cytokeratin fragments from the respectivecytokeratin. Rabbit anti-mouse IgG antibodies coupled to HRP were usedas secondary antibody.

FIG. 3 shows a Western blot on a nitrocellulose filter after SDS-PAGE ofpurified cytokeratin 8 fragmented with chymotrypsin according to theinvention. Each strip of nitrocellulose was allowed to react withdifferent monoclonal antibodies produced as above. A secondary antibody,rabbit anti-mouse IgG coupled with HRP, was added thereafter and thereaction of the monoclonals was visualized with a substrate DAB (SigmaD-5905) according to a known method. The results show that several ofthe monoclonal antibodies react with several of the cytokeratin 8fragments within the size range of about 10 to 50 Kd.

In FIG. 4 there is shown a Western blot on nitrocellulose after SDS-PAGEof purified cytokeratin 18 fragmented with chymotrypsin. Each strip ofnitrocellulose was allowed to react with different monoclonal antibodiesproduced as above. A secondary antibody, rabbit anti-mouse IgG coupledwith HRP, was added thereafter and the reaction of the monoclonals wasvisualized with a substrate DAB (Sigma D-5905) according to a knownmethod. The results show that several of the monoclonal antibodies reactwith several of the cytokeratin 18 fragments within the size range ofabout 10 to 44 Kd.

In FIG. 5 there is shown a Western blot on nitrocellulose after SDS-PAGEof purified cytokeratin 19 fragmented with chymotrypsin. Each strip ofnitrocellulose was allowed to react with different monoclonal antibodiesproduced as above. A secondary antibody, rabbit anti-mouse IgG coupledwith HRP, was added thereafter and the reaction of the monoclonals wasvisualized with a substrate DAB (Sigma D-5905) according to a knownmethod. The results show that several of the monoclonal antibodies reactwith several of the cytokeratin 19 fragments within the size range ofabout 10 to 38 Kd.

All together, the monoclonals show very good reactivity and specificityagainst a majority of fragments from cytokeratin 8 for 7 clones, fromcytokeratin 18 for 7 clones, and from cytokeratin 19 for 5 clones. Thisis in agreement with the specificity of the 15 clones for the wholecytokeratins (see FIGS. 1-2).

Selection of the monoclonal antibodies is made based on the above testsas well as a test in which the antibodies do not react with other humanproteins.

IN VITRO APPLICATIONS OF THE ANTIBODIES ACCORDING TO THE INVENTION

Test for cytokeratin in serum samples from cancer patients and healthypersons

By IRMA and ELISA methods in which monoclonal antibody according to thepresent invention has been coupled to a solid phase (plastic tube) andpolyclonal or monoclonal antibody has been labelled with Iodine-125 orHRP, sera from apparently healthy persons (blood donors) and sera fromcancer patients were tested. As reference and standard material solublecytokeratin fragments according to the invention in buffer solution andhuman serum was used. The levels from the different groups clearlyshowed different quantities (in ng/ml) of cytokeratin fragments.

The results in FIG. 6 show that serum from apparently healthy personsgives a low response in the test, with a very low spread. The majority(95%) of the population falls under 0.9 ng/ml. For serum from themajority (54%) of the cancer patients the values are significantlyhigher.

The above described shows that the present inventors have been able todetect cytokeratin fragments in serum, without any pretreatment thereof,distributed from tumours of cancer patients, using the antibodies andreagents that have been described in the present application. Theantibodies according to the invention are able to react with the mostrepresentative fragments from the above cytokeratins in body fluids ofman. Furthermore, the antibodies according to the present invention areable to react with intact cell and tissue samples. FIG. 7 showscultivated tumour cells (MCF-7) which have been fixed with methanol (toopen up the cell membrane) and thereafter incubated with one of themonoclonal antibodies (M1) according to the present invention, followedby a secondary antibody with FITC coupled thereto. Illumination througha UV light microscope shows the typical cytoskeleton pattern within thecell for several of the cells.

Furthermore, the antibodies of the present invention enableimmunohistology of tissue sections, and immunocytology of, for example,cervix smears, without elaborate pretreatment. FIG. 8 shows animmunohistological section of an adenoma carcinoma of the colon. Thissection is prepared by standard techniques and the cytokeratins in thesample appear by using peroxidase staining. Simple epithelia and tumourcells are revealed.

IN VIVO APPLICATIONS OF THE ANTIBODIES ACCORDING TO THE INVENTION

FIG. 9 shows mice inoculated with DU 145 cells and with developedtumours. The object of this experiment was to show that radioactivelylabelled monoclonal antibodies were able to localize implanted DU 145tumours in mice. The cell line DU 145 has been shown to comprisecytokeratins 8 and 18. (Sherwood E. R., Berg L. A., Mitchell N. J.; TheJournal of Urology, Vol 143, January 1990, pp 167-171).

In blotting of SDS-PAGE, the tested monoclonal antibodies have beenshown to react with the following cytokeratins and their fragments:

    ______________________________________                                        MAb   CYK 8    CYK 18    Fragments 8                                                                             Fragments 18                               ______________________________________                                        6D7   yes      yes (less)                                                                              yes       no                                         2D1   yes      yes (less)                                                                              yes       no                                         3F3   no       yes       yes (less)                                                                              yes                                        ______________________________________                                    

12 mice of NMRI type with an average weight of about 25 g obtained fromBomhults farm, Denmark, were inoculated s.c. with 10 million DU 145cells in RPMI 1640 including 10% FCS. The DU 145 cells were obtainedfrom the research laboratory of the Akademiska hospital, Uppsala,Sweden.

The tumour cells were allowed to grow for 14 days and thereafter themice were randomly divided in 4 groups of 3 mice each.

The monoclonal antibodies and normal mouse IgG (as a control) werelabelled with ¹²⁵ I by using Chloramine-T according to conventionalprocedures.

The respective group was provided with the following radioactivelylabelled antibodies in an amount of 0.3 ml/mouse:

    ______________________________________                                                      Amount of Ab                                                                             Dosis  Spec. Act.                                                                           Conc.                                  Group  Ab     μg      μCi μCi/μg                                                                         μCi/ml                              ______________________________________                                        1      6D7    2.5        12.6   5.0    42                                     2      2D1    2.7        22.5   6.5    75                                     3      3F3    0.71       4.3    6.0    14.2                                   4      XXX    2.8        14.4   5.1    48                                     ______________________________________                                         XXX = Normal mouse IgG                                                   

Scintigraphy for about 20 minutes of choral hydrate anaesthtized micewas performed after 3, 5 and 9 days. After day 9, the mice weresacrified and weight and radioactivity of different organs wasdetermined.

An evaluation of the scintigrammes gives the following results:

    ______________________________________                                        Day     6D7      2D1       3F3   Normal mouse IgG                             ______________________________________                                        3       3/3 ++   2/3 +     2/3 ++                                                                              --                                           5       3/3 ++   2/3 +     2/3 ++                                                                              x                                            9       3/3 +++  1/1 +++.sup.1                                                                           3/3 +++                                                                             --                                           ______________________________________                                         .sup.1 =Two mice died of other reasons                                        X = not done                                                                  -- =no localization of the tumour                                             + = weak localization of the tumour                                           ++ = localization of the tumour                                               +++ = marked localization of the tumour.                                 

FIG. 10 shows scintigraphy of the mice of FIG. 9 showing the in vivolocalizations of the tumours.

FIG. 11 shows an immunoscintigraphy image of a human being. The antibody6D7 is purified and controlled to a quality corresponding to the demandsof drugs. Thereafter it has been labelled with the radioactive isotope I131. Following further purification it was injected into a patienthaving known tumour localizations. The patient was thereafter measuredwith equipment for measuring radioactivity, and the colour scaleindicates the amount of radioactivity: light colour means muchradioactivity and dark colour means no activity. 24 hours after theinjection (24 HPI) there was significantly increased radioactivity onall known tumour localizations and from the image corresponding to theupper part of the chest it clearly appears that even a small metastasein the size range 1 cm can be detected with the radioactive labelledantibody 6D7. The large light portion of the image corresponds to theheart with its contents of blood. Still after 24 hours the bloodcontains some radioactive labelled antibody. Radioactive I 131 falls offthe antibody and is secreted in the urine, and the antibody is degradedby the body to amino acids with time.

The monoclonal antibodies according to the present invention may also beused for in vivo cancer treatment by coupling to cytotoxins orradioactive isotopes, in order to kill tumour cells when the antibodiesare localized in the tumour.

APPLICATION OF THE CYK FRAGMENTS ACCORDING TO THE INVENTION

The cytokeratin fragments according to the invention, besides the usefor production of antibodies, can also be used:

for vaccination of tumour patients, per se or in combination with othertreatment methods.

in kits to perform immunochemical tests, for example ELISA, EIA, IRMA,LIA.

If the fragments are going to be used as a vaccine or antigen/referencematerial in immunological tests, the optional SDS PAGE described aboveis performed and the unwanted effects of SDS are avoided by diluting thefragment solution about 1000 fold in serum or, preferably (especiallyvaccine) in albumin solutions. The addition of proteins also avoidsaggregation of the fragments and provides a "dispersing" effect on theepitopes, with inert proteins. The purification and use of the fragmentsas a vaccine will have to follow the special purity demands of theregistration process in the respective country.

In "sandwich assays" or "double site assays" the cytokeratin fragmentsare used as standard and reference material and the antibodies areeither used as catching antibody coupled to solid phase, e.g. microtiterplates, or as tracer antibody labelled in a suitable way to detect theanalyte, i.e. cytokeratin fragment. In "single site assays" thecytokeratin fragments are coupled to a solid phase and an anti antibodyis used to detect the target substance, i.e. the human antibody againstthe cytokeratin fragment.

The sensitivity of the immunological tests performed with the fragmentsaccording to the invention is shown in FIGS. 12 and 13.

FIG. 12 shows a dose-response curve and the precision profile of anELISA test using the fragments according to the invention asstandard/reference material and the monoclonals coated to the microtiterplates. The sensitivity (calculated as average value+2 SD) is as low as0.1 ng/ml, which proves to be a very high sensitivity.

The same excellent results are obtained in an IRMA test which is shownin FIG. 13.

These sensitive tests are expected to have great applicability inchecking the growth rate in body fluids (without pretreatment) fromepithelial cancer patients and evaluating the effects of monoclonalantibody therapy.

Abbreviations:

ATCC: American Type Culture Collection

cpm: counts per minute

CYK or CK: cytokeratin

DAB: 3,3'-Diaminobenzidine-tetra-hydrocloride

FIA: Freund's incomplete adjuvans

FCA: Freund's complete adjuvans

FCS: fetal calf serum

IRMA: Immunoradiometric assay

ELISA: Enzyme linked immunosorbent assay

LIA: Luminiscence immunoassay

Kd: Kilodalton

HRP: Horse redish peroxidase

FITC: Fluorescein isothiocyanate

PBS-EDTA: Phosphate buffered saline- ethylende diamine tetra acetic acid

RIA: Radio immunoassay

RT: Room Temperature

SDS-PAGE: Sodium dodecyl sulphate-poly acrylamide gel electrophoresis

We claim:
 1. A method of treating epithelial cancer comprisingadministering to a patient in need thereof an effective amount of anantibody selected from the group consisting of an antibody whichspecifically binds to a fragment of cytokeratin 8, an antibody whichspecifically binds to a fragment of cytokeratin 18 and an antibody whichspecifically binds to a fragment of cytokeratin 19, coupled to acytotoxin or radioactive isotope, wherein said antibody is producedbypurifying cytokeratins from epithelial carcinoma cells by preparativeSDS-PAGE; eluting bands corresponding to cytokeratins 8, 18, and 19;digesting said cytokeratins 8, 18, and 19 to produce a mixture offragments ranging in size from 10 to 50 Kd, with the proviso that saidmixture includes fragments other than fragments ranging in size fromabout 38 to 48 kD; immunizing a mouse with a solution comprising saidmixture of cytokeratin fragments; recovering lymphocytes from the spleenof said mouse; fusing said lymphocytes with myeloma cells to producehybridomas; cloning and growing said hybridomas; stabilizing andestablishing single clones of said hybridomas; and recovering amonoclonal antibody which binds to said cytokeratin fragments from saidsingle clones of said hybridomas.
 2. A method of treating epithelialcancer comprising administering to a patient in need thereof aneffective amount of an antibody selected from the group consisting of anantibody which specifically binds to a fragment of cytokeratin 8, anantibody which specifically binds to a fragment of cytokeratin 18 and anantibody which specifically binds to a fragment of cytokeratin 19,coupled to a cytotoxin or radioactive isotope, wherein said antibody isproduced bypurifying cytokeratins from epithelial carcinoma cells;isolating cytokeratins 8, 18, and 19; digesting said cytokeratins 8, 18,and 19 to produce a mixture of fragments ranging in size from 10 to 50Kd, with the proviso that said mixture includes fragments other thanfragments ranging in size from about 38 to 48 kD; immunizing a mousewith a solution comprising said mixture of cytokeratin fragments,recovering lymphocytes from the spleen of said mouse; fusing saidlymphocytes with myeloma cells to produce hybridomas; cloning andgrowing said hybridomas; stabilizing and establishing single clones ofsaid hybridomas; and recovering a monoclonal antibody which binds tosaid cytokeratin fragments from said single clones of said hybridomas.